Method for detecting a substance and microtiter plate

ABSTRACT

The invention relates to a method for detecting an analyte A in a liquid, comprising the following steps: (a) a solution containing an analytical reagent N is provided in a container B; (b) the analyte A is added to the solution, (c) an electrical field eF which acts upon the solution is applied by means of electrodes disposed outside the container B, whereby a modification occurs in the concentration of a substance which is specific with respect to the presence of the analyte A in a region M of the container B and (d) the modification of said concentration is optically detected.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. §371and claims benefit under 35 U.S.C. § 119(a) of International ApplicationNo. PCT/EP02/05910 having an International Filing Date of May 29, 2002,which claims benefit of DE 101 27 045.3 filed on Jun. 2, 2001.

The invention relates to a method for detecting an analyte, and to amicrotiter plate suitable therefor.

The invention relates generally to the area of looking for and detectingpharmacological active ingredients. It is known in the art to bring atest substance or substance into contact with a plurality of differentpotential reactants for example in a microtiter plate. If the substancehas an affinity for a potential reactant, a reaction takes place betweenthe substance and the reactant. The reaction may be, for example, achemical conversion or a binding. The reaction is detected by means of achange in the physical properties of the solution.

Detection methods used are for instance fluorescence polarization,fluorescence resonance energy transfer, fluorescence collerationspectroscopy and radiolabeling methods. The known detection methods arecomplicated. In some cases they require the use of poisonous substances.

Also known in the art are methods which enable a plurality of substancemixtures to be separated in parallel by electrophoresis. For example,Advanced Biotechnologies offers under the sign “Midge” an apparatus withwhich electrophoretic separation of up to 100 DNA samples in parallel ispossible. For this purpose, each DNA sample is felt into a well of agel. An electric field is then applied across the gel so that thesamples are transported into the gel and fractionated there. Evaluationtakes place via the distance the constituents of the DNA sample havemigrated in the gel.—The known method is time-consuming.

DE 199 52 160 A1 discloses a method for detecting a first molecule in asolution, in which a dye-coupled second molecule can bind to the firstmolecule. The net electric charge on the second molecule is in this casesmaller in size than and of opposite sign to the net electric charge onthe first molecule. An anode and a cathode are present in the solution,and an electric field is applied between them. The dye is detectedoptically at the anode if the net electric charge of the first moleculeis negative, and at the cathode if the net electric charge of the firstmolecule is positive. The method requires a complicated measuringapparatus in which a light beam must be focused exactly on the electrodein order to be able to detect the optical change at the electrode.

It is an object of the invention to eliminate the prior artdisadvantages. It is particularly intended to indicate a method and amicrotiter plate with which it is possible universally and without greateffort simultaneously to investigate the reaction behavior of aplurality of substances.

According to the invention, a method for detecting a substance isprovided having the following steps:

-   a) provision of a solution comprising a detection reagent in a    container,-   b) addition of the analyte to the solution,-   c) application of an electric field acting on the solution by means    of electrodes located outside the container, so that the    concentration of an entity which is specific for the presence of the    analyte changes in a region of the container, and-   d) optical detection of the concentration change.

The proposed method is universal. It is simple to carry out. The use ofhazardous or poisonous detection reagents is unnecessary. It is possiblein particular to investigate simultaneously the effect of a large numberof detection reagents on one analyte. The detection reagent is specificfor the analyte. It reacts or binds with the analyte so that itselectrophoretic mobility changes. It is particularly advantageous that,apart from the addition of the analyte in step b, no further addition toor removal from the container is necessary. It is possible thereby toavoid pipetting errors, and a high sample throughput is made possible.Since the electrodes are located outside the container it is possible toavoid electrolytic reactions at the electrodes. A further advantage isthat exact focusing of a light beam on a predetermined point of thecontainer is unnecessary for detection of the concentration change. Theapparatus for optical detection of the concentration change cantherefore have a simpler construction than an apparatus for detectingoptical changes at an electrode.

The solution can be incubated after step b. The container, especiallyits wall or its base, preferably consists at least sectionally of anion-conducting material and the electric field (eF) is applied in step cin such a way that a migration of ions in the material is brought aboutthereby. It is thus possible to achieve a concentration change specificfor the analyte and optically detectable. The ions can moreover migrateout of the liquid into the material, out of the material into the liquidor through the material. No removal of liquid from the container isnecessary for the optical detection of the concentration change in stepd. The optical detection expediently takes place through the openingand/or the base of the container.

The entity is advantageously a detection reagent, a reaction productformed from the detection reagent and the substance, or a competitor.The detection reagent may additionally comprise a receptor, a competitoror a precursor of the reaction product. The receptor is expedientlyselected from the following group: peptide, protein, nucleic acid,sugar, antibody, lectin, avidin, streptavidin, PNA (peptide nucleicacid) or LNA (locked nucleic acid).

The entity may be labeled with a fluorophore. A possible example is amolecular beacon. The entity may be bound onto and/or in the base of thecontainer.

For the optical detection of the concentration change, a light beam ispassed at least through the region, and the change thereof brought aboutby the entity is measured. One possible light beam is a laser beam.Transmitted or reflected light can be utilized. The change brought aboutin the light beam may be a change in intensity, a change in the plane ofpolarization, a scattering angle or the like. The light beam isexpediently guided so that it enters or emerges substantiallyperpendicularly from the base or the opening of the container. Thissimplifies the measurement. The method is thus suitable for standardmicrotiter plate readers.

In a particularly advantageous embodiment, the electric field is appliedsimultaneously across a plurality of containers. The optical detectionof the concentration change in the plurality of containers can likewisetake place simultaneously. The containers are expediently containersarranged in the manner of a microtiter plate on a common support.

Further according to the invention for carrying out the method amicrotiter plate is provided with a plurality of containers which areformed at least sectionally of an ion-conducting material.—The use of anion-conducting material makes it possible to bring about, through amigration of ions which is caused in the electric field, a concentrationchange which is specific for the analyte and which can be detectedoptically.

It is expedient for the walls and/or the base of the containers to beproduced from the ion-conducting material. The base may be produced froman electrical insulator. It may also be activated for the binding of aligand, receptor or substrate. However, it is also possible for areceptor or substrate to be immobilized on the base. In a furtherembodiment, different receptors can be immobilized on predeterminedsections of the base. The base may be produced from glass, quartz orplastic.

The walls may consist of a porous material. They may be activated forthe binding of a ligand. It is additionally possible for the walls tocomprise auxiliaries, e.g. quenchers or protein- or nucleic acid-bindingentities.

The containers may, according to a further embodiment feature, have asubstantially rectangular cross section. In this case two walls of thecontainer are arranged parallel to the electrodes. With such anarrangement, the electric field is developed homogeneously over thevolume of the container.

The ion-conducting material may be produced from a material which ispreferably selected from the following group: agarose, polyacrylamide,cellulose, paper, paperboard, porous silicate, polystyrene, polyvenylchloride, polycarbonate, nylon, polyethylene. Other materials withion-conducting properties are of course also suitable. Theaforementioned materials are expediently in porous form.

In a particularly advantageous embodiment, the containers are in theform of recesses in a first plate produced from the ion-conductingmaterial. The term “plate” is to be interpreted widely in thisconnection. It may also be a sheet or a layer applied to a support. Thefirst plate may be put on a second plate which forms the base, and thefirst plate may produce the walls of the containers. The walls may beformed for example by the inner wall of perforations formed in the firstplate. The second plate may be produced for example from glass or atransparent plastic. According to a further embodiment feature, ahydrophobic covering layer is applied to the first plate. Thisfacilitates the filling of the containers. The hydrophobic material maybe formed from a sheet which is expediently formed from an opaquematerial.

The ion-conducting material may be provided between two electrodes. Theelectrodes may be provided separately from the containers. Theelectrodes may be produced from conventional material such as, forexample, silver, gold, platinum, copper, aluminum or electricallyconducting plastic and the like. They may, for example, be attached tothe ion-conducting material or be in ion-conducting contact therewithvia an aqueous solution which permeates the ion-conducting material.

Exemplary embodiments of the invention are explained in more detailbelow by means of the drawings. These show:

FIG. 1 a diagrammatic cross-sectional view of a first microtiter plate,

FIG. 2 a diagrammatic cross-sectional view of a second microtiter plate,

FIG. 3 a diagrammatic cross-sectional view of a third microtiter plate,

FIG. 4 a diagrammatic cross-sectional view of a fourth microtiter plate,

FIG. 5 a diagrammatic cross-sectional view of a fifth microtiter plate,

FIG. 6 a view from above onto the microtiter plate shown in FIG. 4,

FIGS. 7 a to c a first method variant,

FIGS. 7 d to f a modification of the first method variant,

FIGS. 8 a to c a second method variant,

FIGS. 9 a to c a third method variant,

FIGS. 10 a to c a fourth method variant,

FIGS. 11 a to c a fifth method variant,

FIGS. 12 a to c a sixth method variant,

FIG. 13 a fluorimetric evaluation using a polyacrylamide microtiterplate and

FIG. 14 a fluorimetric evaluation using an agarose microtiter plate.

FIG. 1 shows a diagrammatic cross-sectional view of a first microtiterplate with a particularly simple design. This comprises a first plate 1which has on one side a plurality of recesses 2. Each of the recesses 2forms a container B for receiving a solution containing an analyte. Thefirst plate 1 may consist for example of an agarose or polyacrylamidegel. Such gels can for example be cast in the form of conventionalmicrotiter plates. It is also possible to produce the first plate 1 fromion-conducting material such as cellulose or derivatives thereof. Theshaping can in this case take place by means of compression. The firstplate 1 may moreover be produced from porous polystyrene, polyvenylchloride, polyethylene, polycarbonate, polymethyl metacrylate,polypropylene and the like. It is possible in this case for themicrotiter plate to be produced by the injection molding method.Reference number 3 designates the electrodes attached on the transversesides of the first plate 1. The electrodes 3 can be produced from metalssuitable for this purpose, such as platinum, gold, silver, anelectrically conducting plastic and the like.

In the second microtiter plate shown in FIG. 2, the first plate 1 whichis produced from ion-conducting material is placed on a second plate 4.The first plate 1 has in this case a plurality of perforations 5. Theinner walls of the perforations 5 form the walls of the containers B.The base Bo thereof is formed by the side, facing the first plate 1, ofthe second plate 4. The second plate 4 can be produced for example fromglass, quartz or polystyrene. It is expediently designed to betransparent. The second microtiter plate can be produced in a simplemanner by casting an agarose gel for example on a glass plate. Theperforations in the agarose gel can be produced by suitable plasticcores which have been previously placed on the glass plate and areremoved again after solidification of the agarose or polyacrylamide gel.

For example detection entities such as peptites, proteins, nucleic acidsand the like can be immobilized on the base Bo. It is also possible forthe base Bo to be activated by chemical groups present thereon, such asaldehyde, epoxide, amino groups or biotin. The base Bo may, however,also be produced from an ion-conducting material.

In the third, fourth and fifth microtiter plates shown in FIGS. 3 to 5,in each case a hydrophobic covering layer 7 is applied to the firstplate 1. The hydrophobic covering layer 7 has two perforations 8 whichcorrespond to the first perforations 5. In the fourth microtiter plateshown in FIG. 4, the first plate 1 has neither first recesses 2 norfirst perforations 5. The first plate 1 in this case forms the base Boof the containers B. In the exemplary embodiment shown in FIG. 5, firstrecesses 2 are provided in the first plate 1 and correspond to thesecond perforations 8 in the covering layer 7. The first perforations 5correspond to the second recesses 6, so that recesses 6 form the lowerpart, i.e. a lower wall section and the base Bo, of the containers B.

FIG. 6 shows once again in a view from above the three-layer structureof the microtiter plate of the invention shown in cross section in FIGS.3 to 5. The recesses 2, and first 5 and second perforations 8, can ofcourse also be designed to be rectangular or square.

A subregion of the container B is shown in FIGS. 7 to 12. The containerhas an opening Op at the top and is bounded by wall W and base Bo. Thewalls W and/or the base Bo consist of an ion-permeable material. Thismaterial has the property, on contact with an electrolyte and onapplication of a voltage, of permitting an electrophoretic ion flux intothe material. M designates the region of the container B which isencompassed by an optical measurement. The region M may include thebase. The region M may be, for example, a central region of thecontainer B.

In the first method variant shown in FIGS. 7 a to c, a detection entityN is bound to the base Bo. This may be, for example, a substrate forproteases or other lytic proteins. The detection entity N may be labeledfor example by means of a fluorophore. An analyte is designated with thereference letter A. This may be for example a protease or other lyticproteins. On addition of the analyte A, the detection entity N iscleaved by the analyte and thus a fragment Fr is liberated. Onapplication of an electric field (FIG. 7 c), an electrophoretic forceacts on the fragment Fr and the fragment Fr migrates into or onto thewall W of the container B. The concentration of the fragment Fr in thecontainer B decreases. The fragment Fr has a label, e.g. a fluorophore,or can be detected optically because of other properties. Theconcentration of the fragment can be determined by means of afluorescence measurement in the region M. In this arrangement, thedecrease in fluorescence indicates the present of the analyte A to bedetected.

FIGS. 7 d to 7 f depicts a homogeneous method variant of the methoddescribed in FIGS. 7 a to c. A charged and freely movable detectionentity N is present in a container B. The detection entity N is chargedand may be labeled for example by means of a fluorophore. The detectionentity may be, for example, a substrate for proteases or other lyticproteins. An analyte A is brought into contact with the detection entityN. This may be for example a protease or other lytic proteins. Onaddition of the analyte A, the detection entity N is cleaved by theanalyte A into two fragments Fr1 and Fr2. Fr1 is uncharged and can bedetected optically for example through a fluorophore. Fr2 is charged. Onapplication of an electric field (FIG. 7 f) an electrophoretic forceacts on the detection entity N and the charged fragment Fr2. Detectionentity N and fragment Fr2 migrate into or onto the wall W of thecontainer B. The uncharged fragment Fr1 remains in the container B. Theconcentration of the fragment Fr1 in the container is an indicator ofthe presence of the analyte A. The concentration of the fragment Fr canbe determined by an optical measurement, for example a fluorescencemeasurement in the region M.

In the second method variant shown in FIGS. 8 a to c, a detection entityN is bound to the base Bo of the container B. This is, for example, aligand, an antigen, a receptor, an antibody or a nucleic acid. Ananalyte A is brought into contact with the detection entity N in asolution. The analyte A may be, for example, a receptor, an antibody, aligand, an antigen or complementary nucleic acid. The analyte A binds tothe detection entity N. The analyte A is labeled for example by means ofa fluorogen F. On application of an electric field, the analyte Amigrates into the ion-conducting wall W. If the detection entity N isspecific for the analyte A, part of the analyte A binds to the detectionentity N. The binding of the analyte A to the second detection entity Ncan be detected by means of a fluorescence signal due to the fluorophoreF.

In the third method variant shown in FIGS. 9 a to c, an analyte Adisplaces a detection entity N from its binding to a defined bindingsite. The binding of the analyte A and the binding of the detectionentity N is specific in each case. The binding site may be, for example,an antibody immobilized on the base Bo or an immobilized nucleic acid.The detection entity N may be a specific antigen or a complementarynucleic acid. Displacement of the detection entity N by the analyte Aresults in free mobility of the previously immobilized detection entityN. The detection entity N is charged. Application of an electric fieldmoves the detection entity N into or onto the ion-permeable wall W. Theconcentration of the detection entity N is found by an opticalmeasurement in the region M. The decrease, observable in this case, of apreviously present fluorescence signal is specific for the presence ofthe analyte A in the solution.

In the fourth method variant shown in FIGS. 10 a to c, an analyte A anda charged and freely mobile detection entity N is present in thecontainer B. The analyte A is labeled for example by means of afluorophore. On addition of the analyte A there is a binding, specificfor analyte A, with the detection entity N. Application of an electricfield moves the charged detection entity N and the analyte A boundthereto into or onto the ion-permeable wall W outside the region M. Thedecrease in the concentration of the fluorescent analyte A in thecontainer B can be detected by fluorimetry in the region M.

In the fifth method variant shown in FIGS. 11 a to c, a positivelycharged analyte A and a negatively charged detection entity N is presentin the solution. The analyte A is labeled for example by means of afluorophore. On addition of the analyte A there is a binding, specificfor analyte A, with the detection entity N. Binding of the analyte A tothe detection entity N makes the total charge of the complex zero. Onapplication of an electric field eF, the uncharged complex of detectionentity N and analyte A bound thereto remains in the container B. Unboundanalyte A is moved into or onto the ion-permeable wall W. The diminisheddecrease in the concentration of the fluorescent analyte A in thecontainer B compared with a sample without detection entity N can bedetected by fluorimetry in the region M. The diminished decreaseindicates the presence of the analyte A.

In the sixth method variant shown in FIGS. 12 a to c, the base Bo of thecontainer B is ion-permeable. In addition, a first detection entity N isbound in the base Bo. The base Bo may consist for example of activatedagarose or polyacrylamide to which antibodies or nucleic acids havebeen-bound as detection entity N. A charged analyte A is introduced intothe container B. The analyte A may be labeled for example by means offluorophore. The analyte A comes into contact with detection entity Nthrough diffusion or through application of an electric field eF. Thecontact results in a specific binding between analyte A and detectionentity N. Application of an electric field eF moves unbound analyte Aout of region M, which is detected in an optical measurement. Only thebound analyte A is detected in an optical measurement of the region M.The fluorescence in region M is an indicator of the presence of theanalyte A to be detected.

FIG. 13 shows a fluorimetric evaluation using a microtiter plate of theinvention produced from polyacrylamide. A microtiter plate fluorescencereader is used for the evaluation. Immobilized oligonucleotide A ofsequence 5′-TAA CAC AAC TGG TGT GCT CCT GGA-3′ (SEQ ID NO: 1) waspresent in all the samples 1 to 12. Samples 1 to 4 are in contact withTAMRA-labeled oligonucleotide A′ of sequence 5′-TAMRA-GAG CTA GGA CCTCTT CTG TCC AGG AGC ACA CCA GTT GTG TAA-3′ (TAMRA-SEQ ID NO: 2) which iscomplementary at its 3′ end to the immobilized oligonucleotide A.Samples Nos. 5 to 8 are in contact with TAMRA-labeled oligonucleotide Kof sequence 5′-TAMRA-TAG GGT CAA TGC CAC CCT TTT AAC CTA TCC GGA TTTACG-3′ (5′-TAMRA-SEQ ID NO: 3), which is not complementary tooligonucleotide A. Samples Nos. 9 to 12 merely contained buffer. Thefluorescence is shown here in arbitrary units.

FIG. 14 shows the results of a fluorimetry evaluation using a microtiterplate of the invention produced from agarose. TAMRA-labeledoligonucleotide A′ was present in all the samples. Samples Nos. 1 to 4are in contact with RecA protein. Samples Nos. 9 to 12 contained onlybuffer. The fluorescence is shown in arbitrary units.

The detection can be carried out considerably more quickly by applyingan electric field. The fluorescence can be detected immediately in thepredetermined region of the container. The region may be a centralregion of the container, a region in the vicinity of a wall, or the baseof the container.

EXAMPLES

1. Production of a Supported, Ion-Conductive, Polyacrylamide-BasedMicrotiter Plate with Detection Entities.

A cassette consisting of two glass plates and 1 mm Teflon spacers withthe internal volume 1 mm×80 mm×120 mm was provided on the inner sides ofeach of the glass plates with a plastic support (Gelbond PAG Film,Pharmacia-Amersham), which are suitable for covalent coupling toacrylamide. A plastic support was provided with rows and columns ofsquare recesses which were each 2 mm×2 mm in size and were each 9 mmapart. The recesses were produced with the aid of a cutting blotter(Graphtec). A solution of 20% acrylamide with a monomer to crosslinkerratio of 29:1 was prepared in 1×TBE buffer. Acrydite oligonucleotides A(Eurogentec, Belgium) were added in a concentration of 10 μmol/l asdetection entity before the polymerization. To start the polymerization,70 μl of 10% (w/v) fresh ammonium persulfate and 20 μl of TEMED wasadded, and the solution was poured into the cassette. After one hour,the glass plates were removed and the microtiter plate was placed in aslab electrophoresis chamber with the plastic support with the recess ontop. Unbound charged constituents were removed by subjecting themicrotiter plate to an electrophoresis in 1×TBE at 100 V for one hour.

2. Procedure for a DNA Hybridization Assay Using thePolyacrylamide-Based Microtiter Plate

2 μl portions of sample solution in 1×TBE buffer were put into therecess of the microtiter plate. Four sample solutions in each casecontained:

-   a) 10 pmol of the TAMRA-labeled oligonucleotide A′ prepared by    TibMolbiol, Berlin, with the sequence complementary to    oligonucleotide A.-   b) 10 pmol of the TAMRA-labeled oligonucleotide K, prepared by    TibMolbiol, Berlin, and-   c) no oligonucleotide

Oligonucleotide A′ and K were labeled at the 5′ end with a fluorophore(TAMRA). The microtiter plates were brought into contact with TBE bufferon the sides facing the electrodes, and the microtiter plate wassubjected to an electrophoresis at 50 V for 10 minutes.

3. Fluorimetric Evaluation of the DNA Hybridization Assay

The microtiter plate was inserted into a microtiter plate reader (Lambda320, MWG-Biotech), and the fluorescence of the recess was measured. Thefluorescence was determined with excitation at 540 mm and emission at590 nm in reflected light mode. The fluorescence in the samples witholigonucleotide A′ was distinctly increased compared with thefluorescence of the samples with oligonucleotide B and the pure TBEsample. The increased fluorescence of the sample with oligonucleotide A′indicates the specific binding of oligonucleotide A′ to the Acryditeoligonucleotide A.

4. Production of a Supported, Ion-Conductive, Agarose-Based MicrotiterPlate

Two combs each with four shaping cores of Teflon were suspended 9 mmapart 1 mm above the base in a chamber consisting of a glass plate withtightly surrounding rims of dimension 80 mm×40 mm. The edge length ofthe square shaping cores was 2 mm×2 mm in each case. The shaping coreswere 9 mm apart. A 2% agarose suspension in TBE buffer was heated in amicrowave until the agarose was completely swollen, and about 12 ml ofthe suspension was poured into the chamber. After the agarose hadsolidified, the shaping cores were removed. An arrangement of 2×4recesses with a square opening of 2 mm×2 mm and a depth of about 3 mmhad been produced by means of the shaping cores in the producedmicrotiter plate. An opaque plastic sheet with recesses was placed onthe solidified agarose in such a way that the recess in the sheetcoincided with the recesses in the agarose.

5. Procedure for a DNA-Protein Binding Assay Using the Agarose-BasedMicrotiter Plate.

8 μl of a solution of TBE, 10 μmol/l MgCl₂ and, as detection entity, 10μmol/l oligonucleotide K were introduced into each of the eight recessesin the microtiter plate. Oligonucleotide K was provided with afluorophore (TAMRA) at the 5′ end for labeling. 4 μl of RecA protein(Roche) of a concentration of 1 μg/μl in RecA buffer (10 mmol/l Tris Cl,10 mmol/l MgCl₂, 1 mmol/l DTT, pH 8) was added as sample into each offour recesses. The same volume of RecA buffer without RecA protein wasintroduced as negative control into the remaining four recesses. Themicrotiter plate was incubated at 37° C. for 30 min and then exposed toan electrophoresis at 50 V for 10 min.

6. Fluorimetric Evaluation of the Protein-DNA Binding Assay

The microtiter plate was inserted into a microtiter plate reader (Lambda320, MWG-Biotech) and the fluorescence was measured in the region of therecess. The fluorescence was determined with excitation at 540 mm andemission at 590 nm in reflected light mode. The fluorescence in thesamples with RecA protein was distinctly increased compared with thefluorescence of the samples without RecA protein. The increasedfluorescence of the sample with RecA protein indicates the change in theelectrophoretic mobility of binding of oligonucleotide K owing to thebinding of RecA protein to oligonucleotide K.

LIST OF REFERENCE SYMBOLS

-   1 first plate-   2 first recess-   3 electrode-   4 second plate-   5 first perforation-   6 second recess-   7 hydrophobic covering layer-   8 second perforation-   B container-   Bo base-   A analyte-   N detection entity-   eF electric field-   M region of the container whose optical change is detected-   W wall

The invention claimed is:
 1. A method for detecting the presence of ananalyte in a liquid comprising the steps of: (a) providing a solutioncomprising a detection reagent in a container, wherein the containerconsists of an opening, a wall, and a base, wherein the wall or the baseor a section of the wall or the base consists of an ion-conductingmaterial such that ions can migrate out of the liquid into the materialor through the material when an electric field acting on the solution bymeans of electrodes located outside the container is applied, (b) addingthe analyte to the solution followed by incubating the solution, (c)applying an electric field on the solution by means of electrodeslocated outside the container in such a way that ions migrate out of theliquid into the material or through the material, and the migration ofions causes a concentration change of an entity in a region of thecontainer, wherein the entity is the detection reagent or a reactionproduct formed from the detection reagent and the analyte, and theregion of the container is defined by the opening, the wall and theportion of the base positioned below the opening, and (d) detecting thepresence of the analyte by optically detecting the concentration changeof the entity during or after the applying an electric field of step c),wherein detection is performed through the opening and/or the portion ofthe base positioned below the opening of the container, where a lightbeam is passed at least through the region and where the light beam isguided so that it enters the portion of the base positioned below theopening of the container or the opening, or emerges from the portion ofthe base positioned below the opening of the container or the opening,perpendicularly to the base or the opening.
 2. The method as claimed inclaim 1, where the detection reagent comprises a receptor, a competitor,or a precursor of the reaction product.
 3. The method as claimed inclaim 2, where the receptor is selected from the following group:peptide, protein, nucleic acid, sugar, antibody, lectin, avidin,streptavidin, PNA or LNA.
 4. The method as claimed in claim 1, where theentity is labeled with a fluorophore.
 5. The method as claimed in claim1, where the entity is bound onto and/or in a base of the container. 6.The method as claimed in claim 1, where the electric field is appliedsimultaneously across a plurality of containers arranged in succession.7. The method as claimed in claim 6, where the optical detection of theconcentration change takes place simultaneously in the plurality ofcontainers.